The specific aims of the proposed work are to prepare suitable derivatives of heparin, thrombin and other coagulation proteases as tools to study heparin, antithrombin III and protease equilibirum binding interactions. These interactions will be studied in binary systems to determine affinity constants and stoichiometries of binding. The studies then will be extended to ternary systems to determine whether the binding sites of heparin are specific for antithrombin III and thrombin (or other proteases) or allow competitive protein-heparin interactions. The measurements will be made with heparin isolated both by molecular exclusion chromatography and affinity chromatography on antithrombin III-Sepharose. Binding will be measured both by fluorescence spectroscopy of dansyl derivatives of the heparin or proteases, and by sedimentation equilibrium analysis of heparin-protease complexes. The kinetics of the heparin-catalyzed antithrombin III-thrombin reaction will be studied through the use of the fluorescent, reversible thrombin inhibitor dansylarginine N(93-ethyl-1,5-pentanediyl) amide (DAPA) in order to correlate binding interactions with function. Studies of the binding interaction of Factor Xa also will be undertaken along with investigations of the kinetics of inactivation of Factor Xa by antithrombin III both in the presence and absence of the prothrombinase components Factor Va, phospholipid and Ca++. These studies will provide a model for the modulation of inhibition of coagulation proteases that participate in multicomponent enzymatic complexes. The proposed work should provide further insights into the molecular details of heparin catalysis of the inhibition of coagulation proteases by antithrombin III and the modulation of the coagulation process by heparin. The information gained also should be of value in accomplishing the longer range goal of more generally understanding the regulation of coagulation, and may provide a more rational basis for the therapeutic use of heparin in the control of thrombosis.